This invention relates to reagents and methods for detecting and imaging cardiovascular lesions such as atherosclerotic plaques, vascular clots including thrombi and emboli, myocardial infarction, and other organ infarcts. Monospecific antibody-imaging agent conjugates specific for one type of leukocyte as well as multispecific antibody-imaging agent conjugates specific for at least one type of leukocyte and for antigens associated with fibrin, myosin or platelet cells are used in the present invention. Also used in the present invention are multispecific antibody-imaging agent conjugates specific for at least two different antigens selected from the group consisting of fibrin, myosin, or platelet associated antigens.
When there is an insult to vascular endothelium, circulating blood cells, particularly leukocytes, accumulate. Granulocytes tend to concentrate in the largest numbers, but monocytes and lymphocytes also accumulate to a lesser degree. These cells wander through the vascular endothelium to congregate in the areas of injury. The granulocytes survive in the extravascular space for up to about three days, after which the mononuclear cells, monocytes and lymphocytes, become the dominant population.
Two phases are associated with a vascular insult, a brief early increase in vascular permeability and a more prolonged second phase consisting of increased permeability, attachment of leukocytes, mainly granulocytes, to the vessel wall, diapedesis of predominately leukocytes through the vessel wall, accumulation of leukocytes in the injured area, leukocyte phagocytosis, leakage of fibrinogen and platelets from the vessel, fibrin deposition in the injured area, intravascular clotting with vessel destruction, macrophage engulfment of necrotic debris, migration of fibroblasts and formation of connective tissue, and the neovascularization by ingrowth of capillaries. Thus, the infiltration by leukocytes, particularly granulocytes, is a very early and significant event.
The well-developed atherosclerotic plaque is a result of the interplay of inflammatory and repair events, resulting in a lesion consisting of extracellular calcium salts, cholesterol crystals, glycosaminoglycans, and blood cells and plasma components. Endothelial permeability of arterial walls is induced in early stages of atherosclerosis, allowing the afflux of circulating macromolecules and blood cells, particularly leukocytes (and mainly granulocytes). Secondary changes may involve reduction in permeability of the arterial intima, and the later deposition of platelets and/or fibrin, proliferative, degenerative, necrotic, and repair processes that result in atheromatous lesions. Here again, an early component is the concentration and extravasation of leukocytes in the injured area.
With regard to clots, when vessels are injured, plugging may occur by the formation of fibrin, the aggregation of platelets, and combinations of both. During these events, leukocyte sticking and aggregation, independent of platelet aggregation, occurs. Very early, even before fibrin formation, extravasation of leukocytes takes place.
Deep vein thrombosis (DVT) and pulmonary embolism are very common in the general population, affecting 30% to 60% of otherwise healthy men and women, and up to 80% in high-risk patients. It has been estimated that as much as 20% of all hospital patients are affected with thromboembolic events. In the U.S. alone, it has been estimated that 2.5 million cases occur each year (Sherry, Semin. Nucl. Med. 7: 205-211, 1977).
The majority of commonly used nuclear medicine tests for deep vein thrombosis (DVT) involve nonspecific radiopharmaceuticals employed for radionuclide venography. Thus, there is a great need for a thrombosis-specific radiopharmaceutical for specific, sensitive, and rapid disclosure of thrombi by non-invasive external scintigraphy. Contrast venography, a common radiological method, has been the "gold standard" for DVT, but it has a high incidence of side effects which limit its repeated use (Rabinov and Paulin, Arch. Surg. 104: 134-144, 1972). Compression B-mode ultrasound is also of use for diagnosing the presence of thrombi in the legs, but this is region-limited and, again, not lesion-specific (Lensing et al., N. Engl. J. Med. 320: 342-345, 1989). Hence, radiopharmaceuticals are being sought to achieve simplicity, rapidity, and specificity for the detection and diagnosis of DVT.
Where the aforementioned agents may be useful for DVT, they may fail to disclose pulmonary emboli, which are life-threatening lesions. Different thrombi may require different agents. Venous thrombi consist primarily of polymers of fibrin with entrapped cells, alternating with layers of platelets, whereas arterial thrombi are made up primarily of aggregated platelets with less fibrin (Freiman, in: Coleman et al., eds, Hemostasis and Thrombosis--Basic Principles and Clinical Practice. New York, N.Y., Lippincott, 56: 766-780 (1982)). Thus, there exists a need to have a radiopharmaceutical that can bind to both arterial and venous deposits.
For the most part, the agents available appear to be restricted to either fibrin-directed or platelet-directed pharmaceuticals, as reviewed by Knight (Semin. Nucl. Med., 20: 52-67, 1990).
Fibrin-specific radiopharmaceuticals include radiolabeled fibrinogen, soluble fibrin, antifibrin antibodies and antibody fragments, fragment E.sub.1 (a 60 kDa fragment of human fibrin made by controlled plasmin digestion of crosslinked fibrin), plasmin (an enzyme in the blood responsible for dissolution of fresh thrombi), plasminogen activators (e.g., urokinase, streptokinase and tissue plasminogen activator), heparin, and fibronectin (an adhesive plasma glycoprotein of 450 kDa).
Platelet-directed pharmaceuticals include radiolabeled platelets, antiplatelet antibodies and antibody fragments, anti-activated-platelets, and anti-activated-platelet factors, which have been reviewed by Knight (cited above), as well as by Koblik et al., Semin. Nucl. Med., 19: 221-237 1989), all of which are included herein by reference. Platelet imaging is most useful during the acute phase of thrombosis, when active platelet aggregation occurs, so that these platelet-based imaging methods have difficulty in disclosing clots that are older than 24-48 hours (Oster et al., Proc. Natl. Acad. Sci. USA, 82: 3465-3468, 1985). Another concern is that platelet imaging may be inhibited by concurrent heparin administration in the treatment of these patients (Seabold et al., J. Nucl. Med. 29: 1169-1180, 1988). Heparinization can also reduce the total number of lesions found with anti-fibrin antibodies (Alavi et al., J. Nuci. Med., 29: 825, 1988). In comparison to antifibrin antibodies, fragment E.sub.1 that is radiolabeled appears to demonstrate clots earlier (Koblik et al., cited above). However, the fragment E.sub.1 is difficult to isolate and prepare, and its binding to blood clots is transient (Knight et al, Radiology, 156: 509-514, 1985). A need therefore continues to exist for a preparation containing a combination of two or more of these clot imaging agents, each of which complements the other(s); e.g., an anti-fibrin antibody or antibody fragment or fragment E.sub.1 and an anti-leukocyte antibody or antibody fragment and/or an anti-platelet or anti-activated-platelet antibody or antibody fragment, or an anti-fibrin antibody or antibody fragment and an anti-platelet or anti-activated platelet antibody or fragment; all appropriately labelled.
Inadequate blood and oxygen supply to the myocardium, inducing symptoms of myocardial ischemia or ischemic heart disease, are the usual events resulting from stenotic coronary atherosclerosis. Acute and total coronary artery occlusion results in severe ischemia and, consequently, myocardial infarction. Chronologically, in the first hour, subcellular changes of ischemic heart muscle manifest as mitochondrial granules, reduction of glycogen and respiratory enzymes. Thereafter, from about 1 to 6 hours, margination and clumping of nuclear chromatin, loss of nuclear and myofilament architecture, and infiltration with granulocytes, are observed. In the next phase , from about 6 to 12 hours, typical ischemic necrosis is seen. After 24 hours, severe histological changes are easily seen, leading to focal hemorrhage of different size and dilated capillaries by days 2-4.
The available tests for diagnosing, pinpointing, and determining the extent of myocardial infarction (MI), such as EKG, creatinine kinase (CK-MB) curves, ejection fraction, are all burdened with some limitations. Nuclear imaging methods using .sup.99m Tc pyrophosphate or .sup.201 thallium (TI) have been developed to diagnose and quantify MI. In the last years, antibodies and antibody fragments against myosin have been used experimentally and clinically to demonstrate localization in myocardial cells irreversibly damaged by an ischemic insult (Khaw et al., J. Nucl. Med. 28: 1671-1678 1987); Johnson et al. J. Am. Coll. Cardiol. 13: 27-35, 1989). Uptake of myosin antibody is claimed to be specific for cell death (Framie et al., J. Clin. Invest. 72: 535-544, 1983), and it was found in the clinical studies (cited above) that at least 24 hours are needed before imaging was revealing. Thus, at least 2 or more days were required after the ischemic insult before anti-myosin imaging would work successfully because sufficient cell death must first ensue to result in sufficient antigen sites available for the anti-myosin antibody binding. Thus, there is a need for an agent or combination of agents that will be diagnostic before the occurrence of extensive cell death and myocardial damage and can also combine the attributes of an anti-myosin antibody with a leukocyte-imaging agent. Therefore, for early infarction or even atheromatous plaques, an anti-leukocyte antibody suffices for imaging, such as within the first few hours after the ischemic insult.